Our findings that PlGF is a cancer target and anti-PlGF is useful for anticancer treatment have been challenged by Bais et al. Here we take advantage of carcinogen-induced and transgenic tumor models as well as ocular neovascularization to report further evidence in support of our original findings of PlGF as a promising target for anticancer therapies. We present evidence for the efficacy of additional anti-PlGF antibodies and their ability to phenocopy genetic deficiency or silencing of PlGF in cancer and ocular disease but also show that not all anti-PlGF antibodies are effective. We also provide additional evidence for the specificity of our anti-PlGF antibody and experiments to suggest that anti-PlGF treatment will not be effective for all tumors and why. Further, we show that PlGF blockage inhibits vessel abnormalization rather than density in certain tumors while enhancing VEGF-targeted inhibition in ocular disease. Our findings warrant further testing of anti-PlGF therapies.

Vascular endothelial growth factor (VEGF) and Delta-like 4 ligand (DLL4) are the only genes whose haploinsufficiency results in vascular abnormalities. Although many common pathways are up-regulated in both vascular development and tumor angiogenesis and in vascular remodeling, the role of the Delta/Notch pathway has not been clearly defined in tumor angiogenesis. In this study, we assessed the expression of DLL4, Notch4, and ephrin B2 in transgenic mice developing hepatocarcinoma characterized by a strong remodeling of the tumor sinusoids. We also investigated the role of VEGF in the expression and biological functions of these molecules on human venous endothelial cells. In transgenic livers, we showed that DLL4, active Notch4, and ephrin B2 were gradually up-regulated within the hepatocarcinoma progression and expressed on tumor sinusoidal endothelial cells. In venous endothelial cells, we showed that VEGF up-regulates DLL4 and presenilin, and increased the activation of Notch4, leading to an up-regulation of ephrin B2 with a downregulation of Eph B4. We also showed that the activation of Notch4 is required for VEGF-induced up-regulation of ephrin B2 and the differentiation of human venous endothelial cells in vitro. Accordingly, the disruption of Notch4 signaling by pharmacologic inhibition of presenilin or addition of soluble DLL4 inhibited the effect of VEGF on human venous endothelial cell migration and differentiation. Our study strongly suggests that a coordinated activation of DDL4/ Notch4 and ephrin B2 pathways downstream of VEGF plays a key role in the abnormal remodeling of tumor vessels. (Cancer Res 2006; 66(17): 8501-10)